All human cells possess a cytoskeleton that is key to sustaining stresses and to generating forces as well as to transducing signals. The simplest cell, the Red Blood Cell, has a relatively basic membrane skeleton that is essential to withstanding the stresses of blood flow. The more complex marrow-derived Stem Cells, in comparison, possess a more typical cytoskeleton with a cortex and stress fibers that contribute to attachment as well as probing of the microenvironment in proliferation and differentiation. At least for RBCs, most if not all cytoskeletal proteins are known, and a large number of disease-causing mutants have been identified (involving both protein unfolding and perturbed interactions). Crystal structures are also rapidly emerging for many structural proteins, but cytoskeletal pliability within cells under stress is essentially unknown and likely underlies various pathophysiological processes including some folding diseases. In order to broadly identify cytoskeletal changes in conformation or assembled state directly within stressed cells, we have developed a proteomic-scale approach of in situ labeling of sterically-shielded cysteines with `cell-viable' fluorophores - the method allows multi-level analyses in exploiting fluorescence imaging, quantitative mass spectrometry, and staged multi-dye labeling. Our `Cys Shotgun' studies will significantly improve our molecular understanding in three representative marrow-derived cell systems. We will address in Aim 1 how blood flow stresses affect normal and diseased RBC cytoskeleton, and show with intact membranes that specific domains in spectrin unfold under fluid shear. Preliminary Results indeed show that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin are increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. We will use similar methods in Aim 2 to gain insight into protein conformation and assembly in the two main types of human marrow-derived Stem Cells, particularly focusing on how elastic matrices couple to an active cytoskeleton, affecting expansion & differentiation. Within mesenchymal stem cells, initial results show non-muscle myosin IIA, filamin-A, and vimentin are just a few of the cytoskeletal proteins that exhibit differential labeling in tensed versus relaxed cells. Since red cell membrane diseases and disordered erythropoiesis feature in a number of clinically relevant diseases our findings will likely have significant clinical relevance. These will be the first set of studies to identify protein domains that unfold or dissociate in stressed cells, suggesting specific sites to focus on for stabilization in case of disease. In addition, among the new therapeutic strategies that call for in situ structural insights are exon-skipping strategies that are being applied by others to spectrin superfamily proteins. Methods for identifying cytoskeletal sites of in situ unfolding and deformation should thus provide translational as well as basic insights. PUBLIC HEALTH RELEVANCE: All human cells possess a cytoskeleton that is central to sustaining stresses and generating forces as well as mechano-transducing signals. Red blood cells have a relatively simple membrane skeleton that is key to withstanding the stresses of blood flow; and Stem cells have a more typical cytoskeleton with stress fibers that contribute to actively probing the microenvironment to direct cell lineage. A large number of disease- causing mutants have been identified and some involve protein unfolding. We seek to understand directly within intact cells protein folding/unfolding and association, and we have developed an in situ tagging approach for such purposes. We anticipate our studies will significantly improve our molecular understanding in three representative marrow-derived systems: we will address how stresses affect normal and diseased red cell cytoskeleton, and how elastic matrices couple to an active cytoskeleton to affect stem cell function. Since RBC membrane diseases and disordered erythropoiesis feature in a number of clinically relevant diseases our findings will likely have significant clinical relevance. New therapeutic strategies that could benefit more broadly include exon skipping strategies that are presently being applied to the spectrin family protein dystrophin [Goyenvalle Science 2006].